BIOL 1107 Lab Section ____
19 March 2018
Isolation and Amplification of DNA from E.Coli K12 using PCR and Gel Electrophoresis Analysis
Introduction: In this experiment, my lab group isolated the lacY gene of DNA from an attenuated (weakened in force) strain of Escherichia coli K12 (E.coli) to determine the number of bases through polymerase chain reaction (PCR) and gel electrophoresis. DNA,Deoxyribonucleic acid, is the blueprint for life. It is a a sequence of nucleotides, which is further broken down into base pairs.
During the first section of the lab, the lacY gene of the DNA is extracted from E.coli. First, we used Lysis buffer to denature the cell membrane and open the bacteria cell. Then, the DNA that exits the broken cell is stabilized and cleaned of unwanted protein by adding potassium acetate. Once stabilized, the non-polar solvent, isopropanol, is used to precipitate the negatively charged DNA. Next, we used ethanol to wash the precipitate DNA, and then prevented DNA degradation with nuclease-free water.
During the second section of the lab, PCR is performed to amplify a few copies of a segment of DNA to create thousands of copies. In this lab, the lacY gene of E.coli is amplified. To perform PCR, a target sequence, polymerase, primers, and nucleotides, known as dNTPs are necessary. Primers act as a binding site for the polymerase. Polymerase uses the dNTPs to build a complementary strand of DNA. PCR occurs at three main temperatures. To quickly amplify, it is performed in a thermal cycler to rapidly run through each temperature to denature, anneal, and extend a the lacY gene. Denaturation of the double stranded occurs at 95 °C. This allows for primers to access the sequence and initiate polymerase binding of the now single strand. Primer binding occurs at 60 °C. A primer is a small sequence of single stranded DNA. They complement the sequence on the template DNA to act as a target binding spot for polymerase. Polymerase extends the strand at 75 °C. In a short period of time, the tiny amount of isolated E.coli, lacY, is extended to millions of base pairs, doubling each cycle.
Following, gel electrophoresis is used to find the magnitude, in number of basepairs, in the amplified lacY DNA from the performed PCR. According to the lab manual, gel electrophoresis can be used to separate and identify proteins or DNA fragments based on their size and electrical charge” (Lombard 2017). The amplified lacY DNA is syringed into a well at the top of a electric, agarose, gel field. The side with the wells is a negative cathode and the opposite side is a positive anode. The positive charge attracts the negative DNA strand, and causes it to travel through the gel. The smaller the DNA strand, the greater distance is travels through the agarose gel field. To settle the length of the PCR products, a positive control that produces the expected product, and negative control that does not contain DNA are also syringed into wells for comparison to the lacY sample. I predicted if the lacY gene is accurately isolated and amplified, then the it will be approximately 1,276 base pairs in length. It is proven that the lacY gene amplified during PCR is exactly 1,276 bp in length.
Using the exponential formula of the standard curve in Figure 2 (y = 14123e-0.411x), and the migration distance of the positive control and the PCR products, the size of their bands can be solved. This standard curve equation was formed by plotting the migration distances of the DNA ladder by the number of base pairs that correlate to each band number on a logarithmic semi-log graph (seen in Figure 2). By looking at the gel electrophoresis, the DNA ladder bands can be visually compared to my PCR products to indicate how close they are in length, as well.
Materials & Methods: I followed and used the procedure and materials as stated in the Principles of Biology 1107 Laboratory Manual for Lab 8 and 9 (Lombard 2017).
Freeman, Scott. Biological Science. Pages 189-208. Third edition, Pearson, 2017.
Lombard, Karen . Principles of Biology Biology 1107 Laboratory Manual. Pages 72-81. Fall 2017-Spring 2018, Hayden-Mcneil.
Figures & Tables:
Figure 1, Gel Electrophoresis Results
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